Research ArticleReport of a Novel Mutation in MLH1 Gene ina Hispanic Family from Puerto Rico Fulfilling ClassicAmsterdam Criteria for Lynch Syndrome

Juan M. Marqués-Lespier,1,2 Yaritza Diaz-Algorri,2

Maria Gonzalez-Pons,2 and Marcia Cruz-Correa2,3

1 Department of Medicine, School of Medicine, University of Puerto Rico Medical Sciences Campus, San Juan, PR 00936-5067, USA2University of Puerto Rico Comprehensive Cancer Center, P.O. Box 365067, San Juan, PR 00936-5067, USA3Departments of Medicine, Surgery and Biochemistry, School of Medicine, University of Puerto Rico Medical Sciences Campus,San Juan, PR 00936-5067, USA

Correspondence should be addressed to Marcia Cruz-Correa; marcia.cruz1@upr.edu

Received 25 June 2014; Revised 30 September 2014; Accepted 30 September 2014; Published 20 October 2014

Academic Editor: Antoni Castells

Copyright © 2014 Juan M. Marques-Lespier et al. This is an open access article distributed under the Creative CommonsAttribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work isproperly cited.

In Puerto Rico, colorectal cancer (CRC) represents the second leading cause of cancer in men and women. Familial CRC accountsfor 10–15%of the total CRC cases, while Lynch syndrome accounts for approximately 2–4%of cases. Limited information is availableabout the prevalence, clinical manifestations, and genetic mutations of hereditary CRC inUSHispanic individuals. In this paper wereport a novel mutation in the hMLH1 gene in a Puerto Rican Hispanic family with Lynch syndrome recruited through the PuertoRico Familial Colorectal Cancer Registry (PURIFICAR). Our probandwas identified by applying Amsterdam and Bethesda criteriafor Lynch syndrome, analysis of protein expression by immunohistochemistry, and genetic sequencing of themismatch repair genes.A novel mutation at c.2044 2045 in hMLH1 consisting of the deletion of two consecutive nucleotides (AT) at exon 18 was identified.This deletion causes a frameshift in the protein coding sequence at p.682 resulting in premature termination and a truncatedMLH1protein. To our knowledge, this mutation has not been previously reported in the literature. The detection of this novel mutationin MLH1 further emphasizes the need for genetic testing in at-risk patients for hereditary CRC from various ethnic and racialbackgrounds.

1. Introduction

In Puerto Rico, colorectal cancer (CRC) represents thesecond leading cause of cancer in men and women and thefirst cause of cancer death [1]. In the US, CRC is the thirdleading cause of death in Hispanic population accounting for16.1/100,000 deaths in males and 10.7/100,000 in women [2].Familial CRC accounts for 10–15% of all CRCs [3, 4] andseveral studies suggest that inheritance has a significantimpact in the pathogenesis of up to one third of all CRC cases[5, 6]. One such familial syndrome, accounting for about 1%of all CRC cases, is familial adenomatous polyposis (FAP) [7].Lynch syndrome is a highly penetrant, autosomal dominant

cancer-susceptibility syndrome that accounts for approxi-mately 2–4% of cases of total CRC cases [8, 9]. Lynch syn-drome is caused by germline defects on the DNA mismatchrepair (MMR) mechanism caused by a mutation in one ofthe MMR genes [9]. Individuals with Lynch syndrome aregenetically predisposed to develop malignancies and have a40–80% lifetime risk of developing by age 75 [10–12].

Individuals with Lynch syndrome develop adenomas atthe same rate as the general population, but adenomas tendto develop at an earlier age, have more villous components,and progressmore rapidly [10]. Lynch syndrome is associatedwith tumorigenesis caused by mutations in one of severalgenes involved in DNAmismatch repair system (MMR) [13].

Hindawi Publishing CorporationGastroenterology Research and PracticeVolume 2014, Article ID 527946, 7 pageshttp://dx.doi.org/10.1155/2014/527946

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An additional somatic event in the wild-type allele is neces-sary to make both copies of the MMR gene inactive, whichpredisposes to accumulation of somatic mutations leading tocarcinogenesis. Lynch syndromemutation carriers are also atrisk for other tumors including endometrial cancer and to alesser extent other cancers such as tumors of the stomach,small bowel, ovary, upper uroepithelial tract, biliary tract,skin, and brain [10]. Specific pathological characteristics ofLynch syndrome colorectal tumors have been identified butnone of them are pathognomonic. Mutations leading to thechange of an amino-acid residue in a highly conserved regionof theMMR proteins are usually considered to be pathogenic[14]. Alterations in theMMR genes usually result in the accu-mulation of replication errors, resulting in a hypermutablephenotype known as microsatellite instability (MSI), whichoccurs in a high percentage of Lynch syndrome tumors [9].At present, eight MMR genes are known (hMLH1, hMLH3,hMSH2, hMSH3, hMSH6, hPMS1, hPMS2, and hEPCAM);however, the vast majority of Lynch syndrome cases (90–95%) result from germline hMLH1 and hMSH2 mutations[15].

Due to the limited information available on the preva-lence, clinical manifestations, and genetic mutations in USHispanic individuals with hereditary CRC, variants of uncer-tain significance (VUS) limit the interpretation of genetictesting results among Hispanics who undergo germlinegenetic testing. Published data suggests that amongHispanicsthe majority of MSI CRC tumors may be attributed to Lynchsyndrome, since prevalence of MSI in sporadic colorectaltumors among Hispanic patients is low [2]. Using the PuertoRico Familial Colorectal Cancer Registry (PURIFICAR;http://purificar.rcm.upr.edu/index eng.html) we identifiedpatients meeting Amsterdam criteria and/or Bethesda guide-lines for Lynch syndrome who have had germline genetictesting [16]. The objective of this study was to report a novelMMRmutation found in a Puerto RicanHispanic family withLynch syndrome recruited through the Puerto Rico FamilialColorectal Cancer Registry (PURIFICAR).

2. Methods

2.1. Recruitment. The proband was identified throughPURIFICAR [17]. Individuals with either personal or familyhistory of possible familial colorectal cancer and/or poly-posis are referred to PURIFICARby gastroenterologists, onco-logists, and colorectal surgeons. This registry was establishedin 2006 at the University of Puerto Rico ComprehensiveCancer Center (UPRCCC) and has received direct or in kindsupport from the National Institutes of Health, UPRCCC, thePuerto Rico Gastroenterology Association (http://www.gastropr.org/), and the Puerto Rico Colorectal CancerCoalition (http://cancercolonpr.org/). Subjects enrolled inPURIFICAR are US Hispanic individuals with a clinical and/or genetic diagnosis of FAP, attenuated FAP (AFAP), hama-rtomatous polyposis syndromes, or Lynch syndrome. Con-sented probands complete a comprehensive baseline ques-tionnaire capturing medical, environmental exposures andcancer family history. Pedigrees for each proband are

completed to trace the number of affected relatives withpolyposis and/or cancer using PROGENY software (http://www.progenygenetics.com/) [18]. Pathology reports andmedical/surgical records are obtained to confirm cancerdiagnosis reported by probands. Affected family membersliving in Puerto Rico and US are also invited to participate inthe Registry. In this database, 60% of the probands identifiedare female with an average age of diagnosis of 46.3 (±13.39years). The distribution of the mutations consists of thefollowing: 37.14% in the MLH1 gene, 60% in MSH2, and2.86% in the MSH6 gene. For the current study, analysis waslimited to the proband and his family.

2.2. Patient History. Our proband, defined as the first personin the family inwhich themutationwas detected, is a 63-year-old male with a past medical history of hypertension, hyper-cholesterolemia, and chronic alcohol and tobacco use (23packs a year). He presents with history of recurrentmetachronous colorectal adenocarcinomas.Hewas first diag-nosed at the age of 42 years (1989) with an apple core tumor-ous mass in transverse colon, which was confirmed to beamoderate-poorly differentiated adenocarcinoma.Heunder-went partial colon resection (proximal descending and trans-verse colon) with primary anastomosis and subsequentlyadjuvant chemotherapy. In 2009, at 63 years of age, he under-went surgery after suspecting recurrence of CRC by labo-ratory/imaging studies. A rectosigmoid tumorous mass wasresected and end ileostomy was placed. Pathologic analysisdetermined that the mass was a moderately differentiated,mucin-producing adenocarcinoma that invaded through themuscularis propria into the subserosa with two out ofthree lymph nodes positive for metastasis (T3N1M0). Theproband’s family history was also positive for colorectalcancer: both his mother (diagnosed at the age of 61 years) andsister were diagnosed with colorectal adenocarcinomas. Theproband’s sister, a 58-year-old female with history of hyper-tension, hypercholesterolemia, and chronic smoker (10.5packs a year), was also diagnosed with colon cancer in 2007after presenting with weight loss and lower gastrointestinalbleeding. Her colonoscopy showed a small necrotic ulceratedmass at 70 cm in the proximal transverse colon, whichwas confirmed to be an adenocarcinoma with moderatedifferentiation extending to the muscularis propria withoutinvolving the serosa (T2N1). No evidence of distant metasta-sis was observed by imaging. A detailed pedigree is shown inFigure 1.

2.3. Genetic Analysis. Tumor samples are assigned a uniquebar-code for robotic specimen tracking. Commercialimmunohistochemistry assays for MMR proteins (MSH2,MLH1,MSH6, andPMS2) andmicrosatellite instability analy-sis are performed in all tumor samples byGENZYME [19, 20].Genetic diagnosis of Lynch syndrome was established byidentifying mutations by commercial sequence analyses(MYRIAD Genetic Laboratories, Inc.) [21]. DNA is extractedand purified fromperipheral blood samples or buccalmouth-wash samples, submitted for molecular testing.

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Figure 1: Proband’s Pedigree. Pedigree of the Hispanic family from Puerto Rico fulfilling classic Amsterdam criteria for Lynch syndrome.Exon 18 novel mutation is presented as ∗.

2.3.1. Full Sequence Analysis. Full sequence determinationof MLH1 is performed in both forward and reverse direc-tions of approximately 2,300 base pairs comprising 19 exonsand approximately 560 adjacent noncoding intronic basepairs using fluorescent dye-labeled sequencing primers [21].Aliquots of patient DNA are subjected to polymerase chainreaction (PCR) amplification to generate exon-specific ampli-cons that can be sequenced directly. Electropherogramtracings of each amplicon are analyzed by a proprietarycomputer-based review system followed by visual inspectionand confirmation of all clinically significant variants [21].Genetic variants are detected by comparisonwith a consensuswild-type sequence constructed for each gene. All potentialclinically significant variants are independently confirmed byrepeated PCR amplification of the indicated gene region(s)and sequence determination as described above.

2.3.2. MLH1 Large Rearrangement Analysis. Genomic DNAfrom patients is analyzed by microarray-CGH analysis todetermine copy number abnormalities indicative of deletionor duplicationmutations across theMLH1 gene [21]. Approx-imately 1200 probes have been designed to interrogate all cod-ing exons, limited flanking intron regions, and the respectivepromoters of MLH1 and exons 2-3, 8-9. Approximately 220probes have been designed to interrogate all coding exons.Each probe is analyzed using proprietary software thatcompares the ratio of bound patient DNA to that of a refer-ence DNA to indicate regions of altered copy number. Themicroarray design includes probes to detect deletions andduplications inmultiple genes tested byMGL; however, a datamasking feature is used to limit the analysis only to specificgenes for which testing has been requested. Patient samples

positive for deletions or duplications are confirmed by repeatmicroarray analysis of the genes.

2.3.3. Single Site. DNA sequencing analysis is performed for atargeted gene region containing the specified variant inMLH1. Microarray-CGH analysis is performed for all singlesite large rearrangements inMLH1 [21]. In some cases, long-range PCR analysis and/or sequencing of the resulting PCRproduct is used to detect specific, previously reported inser-tions inMLH1.

3. Results

Our proband’s family history pedigree shows the classicalAmsterdam’s criteria for Lynch syndrome. On the pedigreewe show five family members with history of colorectalcarcinoma, two with concurrent secondary cancers (breastcancer, lung cancer, and endometrial cancer), and one familymember with stomach cancer, which is associated with Lynchsyndrome. In the family line the firstmember to be diagnosedwith any type of cancer is the proband’s grandmother,diagnosed with stomach cancer at 50 years old. In the secondgeneration, we can acknowledge that all of the offspring werediagnosed with colorectal carcinoma. In the third generationcolorectal carcinoma was also diagnosed in three of the fourfamily members including the proband and his sister. A fulldetailed pedigree is shown in Figure 1.

Commercial immunohistochemistry and microsatelliteinstability analysis showed absence of MLH1 and PMS2protein expression and that the tumor was MSI-High. Thesesame analyses were also performed with samples from theproband’s sister with similar results (absence of MLH1 andPMS2 protein expression and MSI-High). Genetic sequence

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ATG1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 18

Stop

ATPase

Exons 13–18Exons 1–11PMS2 binding

Deletionc.2044 2045

Figure 2: Diagrammatic representation of hMLH1 gene with novel mutation. The deletion c.2044 2045 mutation at p.682 described causesa premature stop as highlighted in red. Exons are depicted in blue. The regions encoding functional domains are represented by grey boxes[22].

analysis detected a novel, frameshift mutation at c.2044 2045in exon 18 of the MLH1 gene. A deletion of two consecutivenucleotides (AT) from the positions c.2044 and c.2045 atexon 18 causes a frameshift of the protein coding sequenceat p.682 and a premature termination of the MLH1 proteineleven amino acids away from the frameshift site. This samemutation was also found in the proband’s sister’s daughterand in the proband’s son using single site analysis performedby Myriad Genetic Laboratories [21]. These family membersremain carriers for mutation since neither of them has shownevidence of tumorous lesion in colon or any other regionassociated with Lynch syndrome.The proband also presentedwith a polymorphism in exon 8 of the MLH1 gene. Thisvariant is caused by an A>G change at the nucleotide 655(c.655A>G). This single nucleotide substitution leads to thesubstitution of the isoleucine by a valine at position p.219 intheMLH1 protein.This polymorphism has no known clinicalsignificance.

4. Discussion

Lynch syndrome is a highly penetrant, autosomal dominant-susceptibility syndrome that affects less than 5% of all CRCcases [8, 9]. It is associated with germline mutations in MMRgenes, which increases risk for colorectal and endometrialcancer, as well as gastric, ovarian, urinary, cutaneous seba-ceous glands, and brain cancers [8]. In 1991, the Amsterdamcriteria were developed in order to identify families with thisautosomal dominantly inherited CRC without a polyposisphenotype. However these criteria have limited sensibilityand specificity since 40% of families with known MMRgene mutations do not fulfill the Amsterdam criteria andapproximately 50% of families who meet the Amsterdamcriteria do not have detectable defect in the MMR genes.The most reliable way to diagnose Lynch syndrome is todetect a mutation in the MMR genes in the suspected patient[2]. To date, there are more than 2,400 different MMRgene mutations described throughout the world (http://www.med.mun.ca/MMRvariants/), most of them in theMLH1 andMSH2 genes [22].

Our familial cancer registry, PURIFICAR, contains 35Hispanic families with MMR mutations or MMR proteindeficient tumors. In this registry, we identified our proband, a63-year-oldmale diagnosedwith CRC at age 42.This patient’sfamily history fulfilled Amsterdam criteria for Lynch syn-drome with more than three family members with CRC intwo consecutive generations and more than one familymember diagnosed earlier than the age of 50 years. Also, theproband’s family history presents with multiple cancershighly associated with Lynch syndrome such as endometrialcarcinoma and stomach cancer (Figure 1). The compliancewith Amsterdam criteria and the incidence of Lynch syndr-ome-associated cancers within the proband’s family furthersupport the Lynch syndrome diagnosis.

Upon the evaluation of the proband’s MMR sequenceanalysis, we found a deletion at the level c.2044 and c.2045at exon 18 of the hMLH1 gene.This deletion was subsequentlycomparedwith the available international databases (Interna-tional Society for Gastrointestinal Hereditary Tumors) [23];no report of this deletion was found. Therefore, this deletionin hMLH1may represent a unique mutation in Puerto RicanHispanics. The AT at position c.2044 and c.2045 in exon18 causes a frameshift and subsequently a premature trunca-tion of theMLH1 protein at amino acid 692. It is a deleteriousmutation resulting in themalformation ofMLH1. A diagram-matic representation of hMLH1 gene with novel mutation ispresented in Figure 2.The newly identified mutation causes apremature STOP codon, leading to a nonfunctional protein,which could not be detected by immunohistochemistry anal-ysis. The single nucleotide polymorphism identified in theproband (substitution A>G at the nucleotide 655 (c.655A>G)of the exon 8 of the MLH1 gene), however, is of unknownsignificance and has been described in the literature before[24–27].

The strong history of CRC and Lynch syndrome-associ-ated cancers suggests that the hMLH1 deletion originated inthe maternal line, possibly from the proband’s grandmother.However, the origin of the mutation cannot be definitelydefined sincemost of thematernal family line is deceased andgenetic testing could not be performed. Though, the de novo

Gastroenterology Research and Practice 5

mutation seems to be pathogenic since the deletion waspresent in the subsequent progeny. Similar reports of newlydescribed germline mutations have been described in severalethnic and racial populations [14, 15, 28, 29]. Alvarez et al.described a spectrum ofmutations on Chilean families wherethey also described two unique novel mutations (MLH1intron 15 c.1731 +3 A>T/skipping exon 15; MSH2 exon 13c.2185 2192delATGTTGGAinsCCCTp.M729 E731delinsP729X730) associated with strong Amerindian genetic ancestry[30]. Other highly penetrant mutations have been describedamong Ashkenazi Jews such as the MSH2 c.1906G/C (p.Ala636 Pro), which leads mainly to colorectal cancer [31]. Tworecently published articles by Dominguez-Valentin et al.discussing themutation spectrum of SouthAmerica in Lynchfamilies also showed a high rate of novel mutations in hMLH1and hMSH2 [32, 33]; however neither article presented themutation found in our patient.

To confirm the presence of Lynch syndrome, the MMRgenes of the individuals with MSI tumors are screened forgermline MMR mutations. Similar to the other MMR genes,diverse sequence variants, including nonsense, missense,splice site, and frameshift mutations, have been reportedthroughout the coding region of the MLH1 gene [34]. InPuerto Rico, a population-based study conducted by de Jesus-Monge et al. [35] reported that the prevalence of deficientMMR protein expression (MLH1 and MSH2) among CRCpatients was 4.3% with most cases having absence of MSH2proteins. This study highlights the importance of genetictesting for Lynch syndrome in our Hispanic population andthe need to increase our current understanding of commonmutations and reducing variables of uncertain significance(VUS). VUS are mainly missense substitutions that result insingle amino acid changes but also include in-frame smalldeletions or insertions that change only small numbers ofamino acids, silent coding alterations that may influencesplicing or translation or intronic changes of unknowninfluence on gene splicing. VUS are usually classified as either“pathogenic” or “not pathogenic/low clinical significance”[36]. About 24% of mutations detected in MLH1 are mis-sense substitutions; the majority of these are referred to asunclassified genetic variants [34]. Given that the diagnosisof Lynch syndrome relies primarily on the identificationof germline defects in the MMR genes, detecting theseunclassified genetic variants creates ambiguity in the clinicalsetting, as the pathogenicity of these variants cannot bereadily ascertained [34]. Clinical implications of missense,silent variants, or mutations close to splice site are oftennot clear. Therefore, those variants are classified as VUS andrepresent up to 30% of the identified DNA changes in MMRgenes [37]. Currently the International Society CollaborativeResearch for Gastrointestinal Hereditary Tumors (InSight) istrying to characterize all the missense, deletions, insertions,or any other mutation that may affect MMR genes withthe aim of distinguishing nonpathogenic from pathogenicmutations.

To date, there is very limited information about theprevalence ofMSI amongHispanic CRC patients. Gupta et al.reported in a low prevalence of MSI in the majority studiesincluding Hispanics with CRC, which suggests that MSI

status likely results from germline genetic defects as seen inLynch syndrome [2].This finding emphasizes the importanceof genetic testing in the Hispanic community in order toidentify individuals with Lynch syndrome. Our report of anovel mutation in hMLH1 contributes to the characterizationof pathogenic mutations in the MMR genes leading to Lynchsyndrome.

Core Tip

Very little is known about the prevalence, clinical manifesta-tions, and genetic mutations in US Hispanic individuals withhereditary CRC. Hereditary nonpolyposis colorectal cancer(HNPCC), otherwise known as Lynch syndrome, has beendescribed in up to 5% of CRC cases.This case report presentsa unique mutation in the MLH1 gene in a Hispanic familyfromPuerto Rico leading to a classic case of Lynch syndrome.Our aim is to present this novel mutation to the medicalcommunity.

Conflict of Interests

The authors have no conflict of interests or financial disclo-sure to declare.

Authors’ Contribution

Yaritza Diaz-Algorri and Marcia Cruz-Correa designed thestudy and analyzed the data; Juan M. Marques-Lespierdrafted the paper; Maria Gonzalez-Pons edited the paper.

Acknowledgments

This project is supported by National Institute on MinorityHealth Disparities Award nos. 8U54MD 007587-03 andU54MD007587; NCI Award nos. 5R03CA130034-02 andU54CA096297/CA096300; Center for Collaborative Re-search inHealthDisparities RCMIAwardno.G12MD007600.The authors would like to thank Myriad Genetic Laboratoryfor their support. The authors are grateful to the familymembers for their support and cooperation during this study.

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